1. Field of the Invention
The present invention relates to biotechnology, cellular and molecular biology and, more particularly, to a method for causing photodynamic damage to target cells which may be used in experimental medicine and pharmacology.
2. Description of Related Art
The essence of the photodynamic therapy consists in the introduction to the organism of a photosensitiser (hereinafter referred to as xe2x80x9cPSxe2x80x9d) taken up practically by all cells. Due to differences in microcirculation, distribution, exchange intensity and so on, the PS remains a longer time in target cells, for example cancer cells, than in normal cells. If at that time, target cells and PS molecules occurring therein are exposed to light, the latter shall cause photochemical reactions resulting in the death of target cell.
The main side effect of the photodynamic therapy resides in an enhanced light sensitivity of skin and eyes: patients need to be carefully protected against sunlight within six weeks and more. Another side effect consists in nausea and vomit caused in patients by the introduction of the PS in high amounts (Photodynamic Therapy. Cancer Facts, National Cancer Institute, NIH, CancerNet, 1994).
Both side effects are caused by a combination of two factors: an insufficient PS selectivity as respects target cells, and its excessive concentration in the patient tissues. Improvements in the efficiency of photodynamic damage (hereinafter referred to as xe2x80x9cPDDxe2x80x9d) may be achieved by using the PS as a part of a composition.
There is a prior art composition based on microspheres was abto to enter the cell non-specifically by phagocytosis and penetrate into lysosomes. The application of the compositions of PS with microspheres results in the enhancement of PDD to the cell.
A drawback inherent in compositions with microspheres is that the photodynamic action of these compositions is not specific with respect to a cell type (Bachor, R., Shea, C. R., Gillies, R., and Hasan, T. Photosensitised destruction of human bladder carcinoma cells treated with chlorin e6xe2x80x94conjugated microspheres. Proc. Natl Acad. Sci. USA, 88, 15 80-15 84, 1991).
Another prior art composition comprised chlorin e6 with monoclonal antibodies. This prior art composition contains a component for the target cell identification, a PS carrier component and a PS itself. A disadvantage with such a composition lies in its ability to act on a target cell surface only, wherein PDD induced by the composition does not affect the most PDD-sensitive target cell compartments (Rakestraw, S. L., Tompkins, R. D., and Yarmush, M. L. Antibody-targeted photolysis: In vitro studies with Sn(IV) chlorin e6 covalently bound to monoclonal antibodies using a modified dextran carrier. Proc. Natl. Acad. Sci. USA, 87, 4217-4221, 1990).
This disadvantage is avoided in a composition consisting of a PS, a PS carrier component and a component for target cell recognition and PS transport toward the interior of target cells by specific receptor-mediated endocytosis, said composition being the closest prior art composition to the present invention.
This composition is characterized by the presence of three components performing the same functions: a PS, a PS carrier component and a component for target cell recognition and PS transport toward the interior of the target cells by specific receptor-mediated endocytosis.
An insulin-BSA-chlorin e6 composition, after binding to specific receptors on the surface of target cells, has the ability of receptor-mediated endocytosis and, accordingly, of transport toward the interior of cells where, after cell irradiation, the process of generating active oxygen species takes place, said species being a real cytotoxic agent in inducing PDD to the cell. This composition has an advantage over the aforementioned compositions with monoclonal antibodies or microspheres in that it is capable, through specific binding to cell surface receptors characteristic of this cell type, of being intemalised by receptor-mediated endocytosis owing to which the formation of active photooxidation products occurs inside the cells near to damage-sensitive cell compartments. Although a number of the drawbacks associated with known compositions are removed, the composition lacks any special component to direct the PS transport within of target cells, e.g. transport into the most sensitive cell compartments for TDD. As a result, there is no efficient transport into these compartments, and PDD is not as great as it might be in the presence of said component for directing the transport inside the cells (Akhlynina, T. V., Rosenkranz, A. A., Jans, D. A., Sobolev, A. S. Insulin-mediated intracellular targeting enhances the photodynamic activity of chlorin e6, Cancer Res., 55, 1014-1019, 1995).
As indicated above, side effects are caused by two reasons: an insufficient PS selectivity as respects target cells and their excessive concentration in the patient tissues. Therefore, one of the directions in the development of this subject matter resides in the enhancement to the target cell PDD.
Enhancement of PDD to the target cell, while decreasing an acting PS concentration, is possible by using PS derivatives having other intracellular localisation which are most sensitive to the photodynamic action. As alluded to above, a prior art PDD method employed PS derivatives which penetrate into the cell by non-specific intemalisation and localise in membrane components and lysosomes (Kessel, D. Determinants of photosensitisation by mono-L-aspartyl chlorin e6. Photochem. Photobiol., 49, 447-452, 1989). As also alluded to above, it is also possible to use PS conjugates with microspheres which enter the cell nonspecifically by phagocytosis and penetrate into lysosomes (Bachor, R., Shea, C. R., Gillies, R., and Hasan, T. Photosensitised destruction of human bladder carcinoma cells treated with chlorin e6xe2x80x94conjugated microspheres. Proc. Natl. Acad. Sci. USA, 88, 1580-1584, 1991). Both known methods have the same disadvantage: PS and their derivatives may accumulate both in target cells and normal cells, that is to say, they are non-specific to a cell type (Aizawa, K., Okunaka, T., Kawabe, H., Yasunaka, Y., O""Hata, S., Ohtomo, N., Nishimiya, K., Konaka, C., Kato, H., Hayata, Y., and Salto, T. Localisation of mono-L-aspartyl chlorin e6 (NPe6) in mouse tissues. Photochem. Photobiol., 46, 789-793, 1987).
Improvements in the PDD specificity may be achieved by the PS conjugation with a ligand having specific receptors on the target cell surface. For this purpose, PS compositions with monoclonal antibodies to these cells are proposed. Such an approach enables improvements in the PDD selectivity and efficiency at the expense of a specific recognition of target cells and binding of the compositions to the surface of these cells.
As alluded to above, a prior art method for the target cell PDD comprises a target cell PDD enhancement using PSxe2x80x94chlorin e6 compositions with monoclonal antibodies (Rakestraw, S. L., Tompkins, R. D., and Yarmush, M. L. Antibody-targeted photolysis: In vitro studies with Sn(IV) chlorin e, covalently bound to monoclonal antibodies using a modified dextran carrier. Proc. Nad. Acad. Sci. USA, 87, 4217-4221, 1990): In accordance with this method, a composition comprising the PS is added to target cells, keeping the cell therewith and then exposed to light. Due to the composition properties, the PS is subjected to targeted transport into the cells as a part of the composition, the latter being bound to specific cell receptors. With such a method, plasma membranes are the main target for the PS. This is not optimal, however, since PDD does not extend to include much more sensitive intracellular targetsxe2x80x94the nucleus or lysosomes (Alper, T. Cellular Radiobiology. Cambridge Univ. Press, Cambridge, 1979).
This disadvantage is avoided in the PDD method chosen by the applicant as the closest prior art method to the claimed one.
This method is characterized by the following essential features: a composition for PDD is introduced to target cells; then keeping the cells at a temperature of normal cellular vital activity with the result that target cells uptake a PS as part of the composition; and the PS is subsequently photo-activated (light-irradiated). This method, however, also suffers from disadvantages consisting in that, after cells have uptaken the PS, the latter, owing to the properties of the composition used, follows the way predetermined by the properties of the ligandxe2x80x94a component used for the recognition and receptor-mediated endocytosis of cells. In so doing, it will not necessarily find its way (or finds its way to a slight extent) into other PDD-sensitive cell parts (Akhlynina, T. V., Rosenkranz, A. A., Jans, D. A., Sobolev, A. S. Insulin-mediated intracellular targeting enhances the photodynamic activity of chlorin e6. Cancer Res., 55, 1014-1019, 1995).
It is an object of the present invention to provide compositions and method for causing photodynarnic damage (PDD) to target cells to enable enhancement of target cell PDD owing to a composition capable of effective targeted transport of a photosensitiser (PS) within the target cells, thereby making it possible to improve safety and economy of the method.
This object is solved in a composition producing the target cell PDD in low concentrations due to the fact that the composition, in addition to a PS, a PS carrier component and a component which effects target cell recognition and receptor-mediated endocytosis, also comprises a component capable of effective targeted transport of the PS within the target cells, said composition resulting, due to the PS delivery to the most PDD-sensitive compartments, in the PDD enhancement.